title: Characterization of the 5'-flanking region of the mouse asparagine-linked glycosylation 12 homolog gene.
abstract: Recently, we characterized multiple roles of the endoplasmic reticulum stress responsive element (ERSE) in the promotion of a unique head-to-head gene pair: mammalian asparagine-linked glycosylation 12 homolog (ALG12) and cysteine-rich with EGF-like domains 2 (CRELD2). This bidirectional promoter, which consists of fewer than 400 base pairs, separates the two genes. It has been demonstrated that the ALG12 promoter shows less transcriptional activity through ERSE, but its basic regulatory mechanism has not been characterized. In this study, we focused on well-conserved binding elements for the transcription factors for ATF6, NF-Y and YY1 and the Sp1 and Ets families in the 5'-flanking region of the mouse ALG12 gene. We characterized their dominant roles in regulating ALG12 promoter activities using several deletion and mutation luciferase reporter constructs. The ALG12 gene is expressed in three distinct cell lines: Neuro2a, C6 glioma and HeLa cells. The reporter activity in each cell line decreased similarly with serial deletions of the mouse ALG12 promoter. Mutations in the ERSE and adjacent NF-Y-binding element slightly affected reporter activity. Each of the mutations in the GC-rich sequence and YY1-binding element reduced ALG12 promoter activity, and the combination of these mutations additively decreased reporter activity. Each mutation in the tandem-arranged Ets-family consensus sequences partially attenuated ALG12 promoter activity, and mutations of all three Ets-binding elements decreased promoter activity by approximately 40%. Mutation of the three conserved regulatory elements (GC-rich, YY1 and Ets) in the ALG12 promoter decreased reporter activity by more than 90%. Our results suggest that the promoter activity of the mouse ALG12 gene is regulated in a similar manner in the three cell lines tested in this study. The well-conserved consensus sequences in the promoter of this gene synergistically contribute to maintaining basal gene expression.
authors.affiliation: Department of Biomolecular Science, Faculty of Engineering, Gifu University, 1-1 Yanagido, Gifu, 501-1193, Japan. oohashi@gifu-u.ac.jp
authors.forename: Kentaro
authors.initials: K
authors.lastname: Oh-Hashi
authors.forename: Tomomi
authors.initials: T
authors.lastname: Tejima
authors.forename: Yoko
authors.initials: Y
authors.lastname: Hirata
authors.forename: Kazutoshi
authors.initials: K
authors.lastname: Kiuchi
mesh_terms: D024506:5' Flanking Region / Q000235:genetics*; D051702:Activating Transcription Factor 6 / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D023081:CCAAT-Binding Factor / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000235:genetics; D045744:Cell Line, Tumor; D016326:Extracellular Matrix Proteins / Q000235:genetics; D020862:GC Rich Sequence / Q000235:genetics; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008364:Mannosyltransferases / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D050783:Proto-Oncogene Proteins c-ets / Q000378:metabolism; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012689:Sequence Homology, Nucleic Acid; D016329:Sp1 Transcription Factor / Q000378:metabolism; D050986:YY1 Transcription Factor / Q000378:metabolism
chemical_list: D051702:Activating Transcription Factor 6; D023081:CCAAT-Binding Factor; C561361:CRELD2 protein, mouse; D015815:Cell Adhesion Molecules; D016326:Extracellular Matrix Proteins; D050783:Proto-Oncogene Proteins c-ets; D016329:Sp1 Transcription Factor; D050986:YY1 Transcription Factor; D008156:Luciferases; C586468:ALG12 protein, mouse; D008364:Mannosyltransferases